Bacterial infections remain among the most common and deadly causes of human disease. Unfortunately, the overuse of antibiotics has led to antibiotic resistant pathogenic strains of bacteria. Indeed, bacterial resistance to the new chemical analogs of these drugs appears to be out-pacing the development of such analogs. For example, life-threatening strains of three species of bacteria (Enterococcus faecalis, Mycobacterium tuberculosis, and Pseudomonas aeruginosa) have evolved to be resistant against all known antibiotics. [Stuart B. Levy, "The Challenge of Antibiotic Resistance", in Scientific American, pgs. 46-53 (March 1998)]
Classical penicillin-type antibiotics bind to cell wall synthetic enzymes and thereby deregulate the activity of a single class of proteins known as autolysins which leads to bacterial lysis and bacterial cell death. The development of new drugs which affect an alternative bacterial target protein would be desirable. Pneumococcus is a particularly relevant organism for such study because 1) it has only one autolysin (LytA rather than the multiple autolysins of other bacteria), 2) the autolysin has been cloned and sequenced and can therefore be easily manipulated genetically, and 3) pneumococcus has only one growth zone so that is possible to study activation of the enzyme in a fairly defined region of the cell.
Most bacteria are stabilized by a cell wall consisting of a glycopeptide polymeric murein (peptidoglycan) that completely enclosed the cell [Weidel & Pelzer et al., Enzymol., 26:193-232 (1964)]. Expansion of the cell wall during bacterial growth and splitting of the septum for cell separation requires enzymes that can cleave this covalently closed network. In addition to acting as spacemaker enzymes for cell wall growth [Tomasz et al., Walter de Gruyter, 155-172 (1983)], certain murein hydrolases also act as autolysins, putative suicide enzymes. The life and death dichotomy of autolysin function demonstrates the need for efficient and strict regulation of murein hydrolase activity. Not surprisingly, the regulation of the autolysins is a highly sophisticated physiological task. For example, the enzymes must be controlled at their extracytoplasmic location. In addition, most bacteria possess multiple hydrolases which must be controlled in concert. Antibiotics such as penicillin induce bacteriolysis by interfering with the control of the endogenous autolytic enzymes, indicating the significant chemotherapeutic relevance of these enzymes. Although the binding of antibiotics to cell wall synthetic enzymes has been very well characterized, it is unknown how this event leads to deregulation of autolytic enzymes.
Antibiotic tolerance, a phenomenon distinct from antibiotic resistance, was first described in 1970 in pneumococci and provided a significant clue to the mechanism of action of penicillin [Tomasz et al., Nature, 227:138-140 (1970)]. Tolerance strains stop growing in the presence of conventional concentrations of antibiotic, but do not subsequently die. Tolerance arises when the bacterial autolytic enzymes, i.e., autolysins, fail to be triggered as the antibiotic inhibits the cell wall synthetic machinery. This explicitly implies that penicillin kills bacteria by activating a set of endogenous hydrolytic enzymes and that bacteria exhibit strategies to stop this activation resulting in survival of antibiotic therapy.
Tolerance is of clinical significance since it has been shown that the inability to eradicate tolerant bacteria leads to failure of antibiotic therapy in clinical infections [Handwerger and Tomasz, Rev. Infect. Dis., 7:368-386 (1985); Tuomanen E., Rev. Insect. Dis., 3:S279-S291 (1986); and Tuomanen et al., J. Infect. Dis., 158:36-43 (1988)]. Furthermore, tolerance is thought to be a prerequisite to the development of antibiotic resistance since it creates survivors of antibiotic therapy. These survivors can then acquire new genetic elements of resistance which allow growth in the presence of antibiotics. Virtually all resistant strains also have been shown to be tolerant [Liu and Tomasz, J. Infect. Dis., 152:365-372 (1985)]. Therefore, the identification of novel antibiotics which can lyse these "antibiotic-tolerant" bacteria is necessary.
Mechanistically speaking, tolerance arises in two settings: 1) all bacteria become phenotypically tolerant as growth rate decreases [Tuomanen E., Revs. Infect. Dis., 3:S279-S291 (1986)] and 2) some bacteria are genotypically tolerant by virtue of acquisition of mutations. In both cases, the basic phenomenon is the down regulation of autolysin triggering. This down regulation is transient in phenotypic tolerance in response to environmental cues and is permanent in genotypic tolerance where mutation has changed the lysis control loop. Obviously, the simplest example of genotypic tolerance is the deletion of the autolytic enzymes. This artificial situation was the basis of the first tolerant mutant described in 1970 [Tomasz et al., Nature, 227:138-140 (1970)] but for reasons that are not clear, no clinical isolates have been found which are tolerant because of deletion of these suicidal enzymes. Rather, clinical tolerance arises at the level of regulation of autolysin activity [Tuomanen et al., J. Infect. Dis., 158:36-43 (1988) and Tuomanen et al., Escherichia coli. J. Bacteriol., 170:1373-1376 (1988)].
The most striking examples of powerful regulation of autolysis occur during bacterial response to stress: the stringent response to nutrient deprivation and the heat shock response. The existence of stress-induced global regulators of autolysis described are indicative of strong negative controls on hydrolase deregulation. Thus, bacteria control autolytic activity in order to prevent suicidal lysis. On the other hand, a striking beneficial clinical effect would accrue if one were able to prevent the generation of this protective response in bacteria, particularly in the case of recalcitrant infections involving bacteria sequestered in areas deficient in growth requirements, such as the cerebrospinal fluid, joint fluid, aqueous humor, cardiac vegetations, abscesses, and bone. It stands to reason that the course of therapy for all such infections is prolonged by the need to eradicate phenotypically tolerant bacteria to avoid the rapid relapse observed when antibiotic therapy is withdrawn and surviving bacteria begin to multiply once again. By identifying new antibiotics which can lyse these antibiotic-tolerant bacteria, it should be possible to subvert the protective effects on bacterial survival of slow growth rate or genotypic mutation to tolerance in vivo, thereby globally improving the outcome of antibiotic therapy. Bacteria have developed a complex signaling system that enables the cell to respond swiftly to environmental stress. The histidyl-aspartyl (His-Asp) phosphorelay signal transduction system plays a major role in this signal transduction. There are two key participants in the His-Asp phosphorelay signal transduction system: (1) a sensor histidine kinase, which is generally a transmembrane protein; and (2) a response regulator which mediates changes in gene expression and/or cellular locomotion. The sensor histidine kinase contains a periplasmic or extracellular receptor that detects the external signal, and the sensor histidine kinase then mediates the signal into the cell by activating its corresponding response regulator. The activated response regulator then carries the signal intracellularly to effect the cellular response to the external signal. To date, 23-28 open reading frames have been identified in the Escherichia coli genome as encoding putative sensory histidine kinases, whereas 32 open reading frames have been identified as encoding putative response regulators [Mizuno, DNA Research, 4:161-168 (1997)]. The sensory histidine kinase of the His-Asp phosphorelay signal transduction system contains a specific histidine that is autophosphorylated in the presence of ATP. The sensor histidine kinase transfers the phosphoryl group to a specific aspartyl residue of the response regulator. This phosphoryl transfer activates the response regulator and thereby transduces the signal, allowing the cell to rapidly respond to a particular environmental challenge.
Most bacteria also possess transport ATPases that use the energy derived from their enzymatic hydrolysis of ATP to transport compounds into the cell. In E. coli, for example, the transport ATPases are located in the bacterial inner membrane, and they transport compounds from the periplasmic space into the cell. Transport ATPases are members of a large family of transport proteins termed ABC transporters. The name is derived from a highly conserved ATP-binding cassette contained by all of the members. Generally, ABC transporters are specific for a particular type of molecule (e.g., an amino acid, a sugar, an inorganic ion, a peptide or even a protein). [See, Alberts et al., Molecular Biology of the Cell, 3rd edition, Garland Publishing Inc. (New York) Pages 519-522 (1994)]. Heretofore, the relationship between autolysins, His-Asp phosphorelay systems, and ABC transporters has remained obscure.
Bacteria produce peptides and small organic molecules that kill neighboring bacteria. These bacteriocins are of three varieties based on structure: 1) lantibiotics, 2) nonlantibiotics, and 3) others secreted by virtue of a signal peptide (see Cintas et al., J. Bad., 180:1988-1994 (1998)]. Animals, including insects, also naturally produce peptide antibiotics [Bevins et al., Ann. Rev. Biochem., 59:395-414 (1990)]. These antibiotics have been organized in three structural groups: (1) Cysteine-rich .beta.-sheet peptides; (2) .alpha.-helical, amplipathic molecules; and (3) proline-rich peptides [Mayasaki et al., Int. J. of Antimicrob. Agents, 9:269-280 (1998)]. However, the use of such antibiotics to combat resistant bacterial strains is only beginning to be exploited.
New approaches to drug development are necessary to combat the ever-increasing number of antibiotic-resistant pathogens. In addition, new antibiotics need to be identified which will act independently of autolysins such as the pneumococcal autolysin, LytA. Furthermore, there is a need to provide pharmaceutical compositions containing such new antibiotics in order to more effectively treat bacterial infections and inflammations.
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